Identification of a well-defined and robust cell-culture system for hepatitis C virus (HCV) is critical for facilitating efforts to identify antivirals against HCV. Apath, LLC proposes to develop a series of cell-based assays for testing and screening of compounds with antiviral activity against hepatitis C virus (HCV). These assays will be based on cell lines which contain a constituitively replicating subgenomic HCV replicon. None of the published HCV cell-culture replication systems are sufficiently robust to be used effectively in an antiviral screening assay. Recently, Lohmann et al. described a subgenomic HCV replicon constructed from genotype lb RNA and they isolated Huh7 cell lines which contain this autonomously replicating replicon. Cell lines containing a similar HCV replicon have been made in the laboratory of Charles M. Rice and licensed exclusively to Apath. The cells have been analyzed extensively and have been demonstrated to contain an HCV replicon which exhibits autonomous HCV RNA replication. We propose to maximize the usefulness of these cell lines as drug-discovery tools by formatting assays for compound screening and by modifying the replicon to make the system more amenable to high throughput screening. This system represents the best available system for identifying and testing inhibitors of HCV and thus could be an essential tool for the discovery of new drugs. PROPOSED COMMERCIAL APPLICATION: Apath, LLC proposes to develop a series of cell-based assays for testing and screening of compounds with antiviral activity against hepatitis C virus (HCV). Such are assays critical for evaluating lead compounds with biochemically identified activity against HCV and also to assist in efforts to identify new antivirals for treatment of HCV infections.